BioRelianceBiologics Safety Testing Services
Pinnacle Q-PCRTM Mycoplasma and Spiroplasma Detection Assays (EP
2.6.7) IntroductionNucleic acid amplification (NAT) methods for the
detection of mycoplasma and spiroplasma species using real time
Q-PCR can offer rapid turnaround to result. The Pinnacle Q-PCRTM
platform developed at BioReliance couples state of the art
bioinformatics for optimal assay design with automated sample
preparation and assay set up. These features make the Pinnacle
Q-PCRTM Mycoplasma detection assay an ideal next-generation test to
augment current microbiological methods.
Mycoplasma testing is governed by regulatory guidelines EP, USP,
PTC, and JP. Recently the use of NAT test methods for the detection
of mycoplasma has been described within the FDA guidance for
Industry and WHO technical series report series as well as
European, United States and Japanese Pharmacopoeia. These reports
have provided support for NAT as a suitable alternate to
conventional test methods when appropriately validated.
BioReliance has developed an NAT based test method that couples
automated DNA extraction with auto-mated real time Q-PCR detection
to provide a rapid and validated test for the detection of
Mycoplasma and Spiroplasma species.
Assay method for detection of Mycoplasma and Spiroplasma species
using Pinnacle Q-PCRTMThe efficiency of the extraction system is
critical for the ability to support the introduction of a real-time
Q-PCR assay for detection of Mycoplasma and Spiroplasma species
which meets the European Pharmacopoeia regulatory guidelines (EP
2.6.7). The extraction system developed for this assay utilizes
increased volumes of test material, followed by steps to
concentrate the Mycoplasma/Spiroplasma species, then processing on
an automated nucleic acid purification platform. Further
purification of the nucleic acid renders the material suitable for
evaluation in the Pinnacle Q-PCRTM system.
Fully automated liquid handling systems then prepare the 384
well PCR plates. All amplification and analysis is carried out on
fully validated real-time PCR amplification and detection
equipment.
Nucleic acid amplification technology is then used to
exponentially increase the amount of target sequence that may be
present in the DNA extract. The test system employs real time PCR
technology to detect the presence of target sequences within a test
sample.
Rapid real-time PCR (Q-PCR)
based method for detection of
Mycoplasma and Spiroplasma
species
Pinnacle Q-PCRTM System
incorporates advanced assay
design with automation
to ensure sensitivity and
reproducibility
Fully validated, regulatory
compliant test method
Amplification plot of Mycoplasma orale plasmid positive control
spanning a concentration of 1 × 108 to 1 × 101 copies per
reaction.
Standard curve plot of cycle threshold vs concentration which
illustrates a linear relationship over the concentration range of 1
× 101 to 1 × 108 copies per reaction.
www.bioreliance.comNorth America Toll Free: 800 553 5372 Tel:
301 738 1000
Europe & International Tel: +44 (0)141 946 9999 • Japan Tel:
+81 (0)3 5796 7430Email: [email protected]
©2012 Sigma-Aldrich Co. LLC. All rights reserved. BioReliance
and SAFC are trademarks of Sigma-Aldrich Co. LLC or its Affiliates,
registered in the US and other countries. F-0790811
BioRelianceBiologics Safety Testing Services
The test employs three independent PCRs• Detection of
mycoplasma• Detection of spiroplasma• Detection of EPC (Extraction
Positive Control) internal control
Parameter BioReliance Pinnacle Q-PCRTM Conventional methods
Method Real-time PCR Agar and broth amplification; indicator
detection system
Endpoint Fluorescence Mycoplasma colony count
AppropriatenessIn Process Testing, Raw Materials, Cell Bank
Screening, Investigations
Designed for Lot Release Testing. Also used for Raw Material
Testing.
Sensitivity 10 cfu/ml† 10-100 cfu/ml
Assay Duration 5 hours 28 days
Specificity High High
Validation Yes Yes
Range of Species 102 species* Viable
Robustness Yes Yes
Scalability Yes Yes
Regulatory ICH Guidelines EP/USP compliance EP/USP/PTC
compliant
*Validated against Mycoplasma. orale, Mycoplasma hyorhinis,
Mycoplasma synoviae, Mycoplasma fermentans, Mycoplasma arginini,
Mycoplasma pneumoniae, Acholeplasma laidlawii, Spiroplasma
citri.
† Exceeds the 100cfu/ml requirement for non-cultivable
(indicator cell culture method) Mycoplasma hyorhinis.
Ordering Information
Assay Number Assay Description Regulatory Compliance Sample
Requirements
300200GMP.BUK Rapid Pinnacle Q-PCRTM Mycoplasma Detection Assay
(EP 2.6.7) (3 day turn-around time)
EP/USP, GMP 2 × 2 ml or 2 vials 5 × 105 cells/ml
300201GMP.BUKPinnacle Q-PCRTM Mycoplasma Detection Assay (EP
2.6.7) (7 day turn-around time)
300202GMP.BUKRapid Pinnacle Q-PCRTM Mycoplasma and Spiroplasma
Detection Assay (3 day turn-around time)
EP/USP, GMP 4 × 2ml or 4 vials 5 × 105 cells/ml
300203GMP.BUKPinnacle Q-PCRTM Mycoplasma and Spiroplasma
Detection Assay (7 day turn-around time)
ReferencesWorld Health Organization (2010). Recommendations for
the evaluation of animal cell cultures as substrates for the
manufacture of biological medicinal products and for the
characterization of cell banks. Proposed replacement of TRS 878,
Annex 1. Adopted October 2010.
European Pharmacopoeia, Chapter 2.6.7. Mycoplasmas.
01/2008:20607.
United States Pharmacopoeia 34, General chapter 63, Mycoplasma
Tests August 1, 2011.
Japanese Pharmacopoeia XV, 14. Mycoplasma testing for Cell
Substrates used for the Production of Biotechnological/Biological
Products. Supplement II. October 1, 2009.
PDA Technical Report No. 50, Alternative Methods for Mycoplasma
testing. 2010.
